"Bioengineered DNA was, weight for weight, the most valuable material in the world. A single microscopic bacterium, too small to see with the human eye, but containing the gene for a heart attack enzyme, streptokinase, or for "ice-minus" which prevented frost damage to crops, might be worth 5 billion dollars to the right buyer."

Michael Crichton - Jurassic Park

INTRODUCTION

DNA (deoxyribonucleic acid) is the genetic code responsible for giving organisms certain phenotypes. One of the basic tools of modern biotechnology is DNA splicing, cutting DNA and linking it to other DNA molecules. The basic concept behind genetic engineering is the process of removing a functional DNA fragment - a gene - from one organism and combining it with the DNA of another organism in order to make the protein that the gene codes for. For example, currently some plants are genetically engineered in that they acquire genes for resistance to pests or diseases. Also, in the cases of gene therapy for humans, functional genes can be given to people with non-functional or mutated genes, such as in a genetic disease like cystic fibrosis.

In this laboratory experiment, students gain a hands-on experience with the technique of genetic engineering. A non-pathogenic strain of E.coli bacteria is mixed with a plasmid that contains the Lux gene and a plasmid that contains the GFP gene, respectively. A plasmid is a small, circular piece of DNA typically allowing a cell to become resistant to an antibiotic and allowing the synthesis of some other gene of interest. The Lux gene is a Luciferase gene that has been isolated and purified from a glowing bacteria, Vibrio fischeri, but is similar to the firefly gene responsible for its "glowing in the dark" phenotype. On the other hand, the GFP gene is a Green Fluorescent Protein gene that has been isolated and purified from the bioluminescent (fluorescent) jellyfish, Aequoria victoria.

Transformation occurs when a cell takes up and expresses added DNA. When competent E.coli cells are successfully transformed with these two different plasmids, they acquire an additional trait. The E.coli cells transformed with the pLux will glow in the dark, whereas the E.coli cells transformed with the pGFP will fluoresce under a long wave UV light.

This genetically engineered plant Glows-in-the-Dark!

This laboratory experiment is an excellent opportunity for the issues regarding the current and the future genetic engineering of plants, animals, and eventually HUMANS to be discussed. In the very near future, the Human Genome Project will be completed and therefore, the genes that code for every aspect of a human being will be identified. The impact of this knowledge on society as a whole can be discussed :

Should we have the right to genetically engineer ourselves "for the better" or to prevent illnesses?

Should we have the right to genetically engineer our children?

Will only the rich benefit from this?

Will this generate "segregation" in our world - the genetically engineered people and the non-genetically engineered people?

A genetically engineered mouse that Glows-in-the-Dark!

OBJECTIVES

Transform bacterial cells with a plasmid and observe the growth of the bacteria and the genetically engineered phenotypic traits. The plasmids used will include some of the following:

pLux: allows resistence to ampicillin and allows bacteria to GLOW IN THE DARK.
pGFP: allows resistence to ampicillin and will make the bacteria fluoresce green under a "blacklight".

LB/AMP (Luria Broth with Ampicillin) plates (3 per group - 1 for pLux, 1 for pGFP, and 1 for Control {no DNA}

37oC Incubator

Long wave UV lights

Straws

LB (Luria Broth) - 1 ml. aliquots per group

10-100 ul. capacity micropipettors

PROCEDURES

Instructor: The instructor will prepare competent bacterial cells. Bacterial cells normally will not take up plasmid DNA unless conditioned to do so. The following procedure will be used to prepare competent cells: